Molecular Mechanism of Metal-Independent Decomposition of

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Molecular Mechanism of Metal-independent Decomposition of Organic Hydroperoxides by the Halogenated Quinoid Carcinogens and the Potential Biological Implications Chun-Hua Huang, Fu-Rong Ren, Guo-Qiang Shan, Hao Qin, Li Mao, and Ben-Zhan Zhu Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/tx500486z • Publication Date (Web): 19 Mar 2015 Downloaded from http://pubs.acs.org on March 22, 2015

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Chemical Research in Toxicology

Molecular Mechanism of Metal-independent Decomposition of Organic Hydroperoxides by the Halogenated Quinoid Carcinogens and the Potential Biological Implications Chun-Hua Huang†#, Fu-Rong Ren†#, Guo-Qiang Shan§, Hao Qin†, Li Mao†, Ben-Zhan Zhu†*

†

State Key Laboratory of Environmental Chemistry and Ecotoxicology

Research Center for Eco-Environmental Sciences, CAS Beijing 100085, China. §

* [email protected]

Key Laboratory of Pollution Processes and Environmental Criteria, MOE,

Nankai University, Tianjin 300071, China #

Contributed equally

ACS Paragon Plus Environment


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TOC

O Cl

Cl O

O

O OR

ROOH

HCl

Cl O Cl

O

O

OOR Cl

O

O Cl

O

O


O

OH OR

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Abstract: Halogenated quinones (XQ) are a class of carcinogenic intermediates and newly-identified chlorination disinfection byproducts in drinking water. Organic hydroperoxides (ROOH) can be produced both by free radical reactions and enzymatic oxidation of polyunsaturated fatty acids. ROOH have been shown decompose to alkoxyl radicals via catalysis by transition metal, which may lipid peroxidation or transform further to the reactive aldehydes. However, it is clear whether XQ react with ROOH in a similar manner to generate alkoxyl radicals

metal-independently.

By

complementary

applications

of

ESR

spin-trapping, HPLC/high resolution mass spectrometric and other analytical methods,

we

significantly

found

that

enhance

the

2,5-dichloro-1,4-benzoquinone decomposition

of

a

(DCBQ) model

could ROOH

tert-butylhydroperoxide, resulting in the formation of t-butoxyl radicals independent of transition metals. Based on the above findings, we detected and identified, for the first time, an unprecedented C-centered quinone ketoxy radical. Then we extended our study to the more physiologically relevant endogenous ROOH 13-hydroperoxy-9,11-octadecadienoic acid, and found that DCBQ could also markedly enhance its decomposition to generate the reactive lipid alkyl radicals and the genotoxic 4-hydroxy-2-nonenal (HNE). Similar results were observed with other XQ. In summary, these findings demonstrated that XQ can facilitate ROOH decomposition to produce reactive alkoxyl, quinone ketoxy, lipid alkyl radicals and genotoxic HNE via a novel metal- independent mechanism, which may explain partly their potential genotoxicity and carcinogenicity.

Key Words: Halogenated quinones; Hydroperoxides; Free radicals; Quinone ketoxy radical; ESR spin-trapping



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Abbreviations XQ, Halogenated quinones; ROOH, Organic hydroperoxides; DCBQ, 2, 5-dichloro-1,4-benzoquinone; t-butoxyl radicals;

t-BuOOH,

tert-butylhydroperoxide;

t-BuO•,

CBQ-O•, 2-chloro-5-hydroxy-1,4-benzoquinone radical;

•

CBQ=O, carbon-centered quinone ketoxy radical;


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1. Introduction Halogenated quinones (XQ) are a class of toxic intermediates that can initiate many harmful effects in vivo, such as acute hepatoxicity, nephrotoxicity, and carcinogenesis.1,2 Chlorinated 1, 4-benzoquinones (such as tetrachloro- and 2,5-dichloro-1, 4-benzoquinone) are the major genotoxic and carcinogenic quinoid metabolites of the widely used pesticides chlorinated phenols (the wood preservative pentachlorophenol (PCP) and 2,4,5-trichlorophenol). PCP has been detected in at least 1/5 National Priorities List sites recognized by the US EPA and classified by the IARC as a group 2B environmental carcinogen.3 XQ have also been identified as toxic intermediates or products during the oxidation or destruction of halophenols and other polyhalogenated persistent organic pollutants (POPs).3-6 Recently, a dozen of XQ were found to be new chlorination byproducts in disinfected drinking water and in swimming pool waters,7,8 and some were also identified in pulp and paper mill discharges. These XQ were found to cause damage to DNA and protein by both oxidation and adduct formation,1-6, 9-11 and therefore they are potentially carcinogenic. Organic hydroperoxides (ROOH) can be produced both non-enzymatically and enzymatically.12,13 ROOH have been shown to decompose to alkoxyl radicals (RO•) via transition metal catalysis, which may cause de novo lipid peroxidation or further transform to α, β-unsaturated aldehydes that can react with and damage DNA, proteins and lipids.12,13 ROOH + Me(n-1)+ → RO• + OH- + Men+

Reaction 1

We have shown recently that hydroxyl radical and chemiluminescence can be produced during the decomposition of H2O2 by tetrachloro-1,4-benzoquinone (TCBQ) and other XQ (Fig. 1) in a metal-independent manner, and a novel nucleophilic substitution coupled with homolytical decomposition mechanism was proposed (Fig. 2).14-18 Potent oxidative DNA damage (8-oxodeoxyguanosine formation) and methyl oxidation of 5-methyl-2’-deoxycytidine by XQ and H2O2 were observed via a



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metal-independent hydroxyl radical mechanism.19-21 However, it is unclear whether XQ interact analogously with ROOH to generate RO• transition metal-independently. Therefore, in a recent study we tried to address these questions. (i) Can the ROOH decomposition be facilitated by XQ to generate RO•? (ii) if so, is RO• generation metal-dependently? (iii) and what is the underlying molecular mechanism?

2. Metal-independent ROOH decomposition and RO• generation by XQ Using 2, 5-dichloro-1,4-benzoquinone (DCBQ) as a model XQ, and tert-butylhydroperoxide (t-BuOOH) as a model ROOH, we found that DCBQ could markedly enhance t-BuOOH decomposition and the generation of the DMPO adducts with t-butoxyl radicals (t-BuO•) and methyl radicals (•CH3) (Fig. 3).22 It is worth noting that a small radical signal was also detected in both DCBQ and 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ) systems, but could not be observed in Fe and other XQ systems (for further identification of this radical adduct, see below Section 3). In contrast, incubation of either compound alone did not lead to t-BuO• or •

CH3 generation (Fig. 3). The middle triplet signal for DCBQ alone was found to be

the 2,5-dichloro-semiquinone anion radical (DCSQ•-). Together, the above findings indicate that DCBQ can facilitate t-BuOOH decomposition and the generation of t-BuO• and •CH3. Redox-active transition metals, especially Fe and Cu, have been found to mediate t-BuO• and •CH3 generation from t-BuOOH.12,13 Therefore, several specific metal chelators for Fe and Cu were employed to study the potential role of trace amount of such metal ions.12, 23-25 Neither DMPO/t-BuO• nor DMPO/•CH3 signal was affected by the addition of several Fe(II)-specific or Cu(I)-specific chelators. These findings clearly indicate that transition metal ions contaminated in the system are not responsible for the radical formation. The metal-independent t-BuO• and •CH3 generation was not restricted to DCBQ and t-BuOOH, but was also found when DCBQ was replaced by other XQ. However, no t-BuO• and •CH3 generation was observed from t-BuOOH and the non-XQ, or the methyl-substituted quinones.


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It should be noted that DCSQ•- was detected once DCBQ was put to phosphate buffer. Water was suggested as the major source of the electron to reduce DCBQ via hydrolysis of DCBQ.26 DCSQ•- signal was found to be remarkably reduced after t-BuOOH addition, with concurrent t-BuO• generation (c.f. Fig. 3). These results indicate that DCSQ•- may directly reduce t-BuOOH to t-BuO• (Reaction 2), which is similar to the Fe(II)-catalyzed t-BuOOH decomposition to t-BuO• (Reaction 3):

t-BuOOH + DCSQ•- →

t-BuO • + OH- + DCBQ

Reaction 2

t-BuOOH + Fe2+ → t-BuO • + OH- + Fe3+

Reaction 3

If the above mechanism were true, then the generation of t-BuO• from t-BuOOH/DCBQ should be dependent on DCSQ•- concentration, i.e., the higher the concentration, the more t-BuO• will be generated; and the main product of this reaction should be DCBQ. However, no DMPO/t-BuO• was observed from the reduced form of DCBQ, 2,5-dichlorohydroquinone (DCHQ) and t-BuOOH, although high DCSQ•- concentrations were generated during DCHQ auto-oxidation (Fig. 3). Interestingly, DMPO/t-BuO• could be observed again when DCHQ was quickly transformed by myeloperoxidase to DCBQ (Fig. 3). In addition, DMPO/t-BuO• formation was observed to be directly dependent on DCBQ concentration. These findings indicate that DCBQ, rather than DCSQ•-, may be critical for t-BuO

•

generation. Electrospray

ionization

quadrupole

time-of-flight

mass

spectrometry

(ESI-Q-TOF-MS) was then employed to characterize the final reaction product for DCBQ/t-BuOOH,

which

was

identified

as

the

ionic

form

of

2-chloro-5-hydroxy-1,4-benzoquinone (CBQ-OH). Therefore, the metal-independent t-BuO• generation by DCBQ/t-BuOOH seems not to occur via a DCSQ•--mediated Fenton-like reaction. On the basis of the above findings, we proposed a novel mechanism for DCBQ-enhanced t-BuOOH decomposition and t-BuO• and •CH3 generation: t-BuOOH may first attack DCBQ via nucleophilic

substitution,

generating

an

unstable


chloroquinone/peroxide


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CBQ-OO-t-Bu,

which

decompose

homolytically

to

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forn

t-BuO•

and

an

oxygen-centered quinone enoxy radical (CBQ-O•) (see Fig. 4).22

3. Characterization of a novel carbon-centered quinone ketoxy radical intermediate In the above studies, we found that XQ could enhance ROOH decomposition and alkoxyl radical generation via a metal-independent mechanism.22 However, both the major reaction products, and the suggested chloroquinone/peroxide intermediate CBQ-OO-t-Bu and enoxy radical CBQ-O• were unequivocally charaterized. Therefore, in the following study semi-preparative HPLC method was employed in order to swiftly obtain the hypothesized reaction intermediate and major products.27 Expectedly, one of the major products was found to be CBQ-OH. Another product was first assumed to be the CBQ-OO-t-Bu intermediate (MW 230), since this product was identified by ESI-Q-TOF-MS with 1-Cl isotope clusters at m/z 229, and MS/MS study found that it could be fragmented easily to form a peak at m/z 172. However, further spectral investigations suggested that it was not the intermediate CBQ-OO-t-Bu, since it is a rather stable compound which may contain an OH-group. Finally, it was identified, unexpectedly, as the rearranged isomer of the intermediate CBQ-OO-t-Bu,

2-hydroxy-3-t-butoxy-5-chloro-1,4-benzoquinone

(CBQ(OH)-O-t-Bu) (for its chemical structure, see Fig. 4). Two new issues arise from this unexpected result: 1) Is the new product CBQ(OH)-O-t-Bu directly resulting from the unstable CBQ-OO-t-Bu; and 2) If so, what is the exact mechanism? To address these issues, a new hypothesis was proposed: The unstable CBQ-OO-t-Bu may undergo homolytical decomposition, resulting in t-BuO• and CBQ-O• generation; The O-centered CBQ-O• might be readily isomerized to its corresponding more stable C-centered ketoxy radical (•CBQ=O). The coupling of •CBQ=O with t-BuO• should generate CBQ(OH)-O-t-Bu (see Fig. 4). If the above assumption were right, DMPO should inhibit CBQ(OH)-O-t-Bu formation since it can compete with •CBQ=O to trap t-BuO•. We found that this was exactly the case.


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Interestingly and unexpectedly, a new peak was detected from DCBQ/t-BuOOH with DMPO, which was identified by ESI-Q-TOF-MS with 1-Cl isotope peak clusters at m/z 268. These findings suggest that it might be a DMPO adduct with either CBQ-O• or •CBQ=O radical (here simply referred as DMPO-157). Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was then used to obtain more accurate information of DMPO-157.28,29 DMPO-157 was identified by FTICR/MS with 1-Cl isotope peak clusters at m/z 268.0383, corresponding to the nitrone form of DMPO-157. When t-BuOOH was substituted by H2O2 and cumene hydroperoxide, the same DMPO-157 was detected. In our earlier DMPO spin-trapping study on DCBQ/t-BuOOH, a minor DMPO adduct with weak ESR signals was also detected, but not identified yet (see Fig. 3).22 With the newly-obtained FTICR/MS results, we speculated that this unknown radical signal might be the DMPO adduct with either enoxy or its ketoxy radical. However, due to the strong disturbance by the ESR signals from other DMPO radical adducts, only a weak 4-line ESR signal with equal intensity could be detected. Therefore, it is unclear just from these findings whether the DMPO trapped radical is an O- or a C-centered quinone radical. To avoid the strong interference of other ESR signals of DMPO radical adducts and get better DMPO-157 ESR signal, we investigated DCBQ/H2O2 with DMPO. Beside the expected DMPO/•OH, we also detected a 6-line ESR spectra with the same intensity (aH = 28.8 G; aN = 17.0 G; aN /aH = 0.59) (Fig. 5), which are characteristic of the spin-trapping of a C-centered •CBQ=O, rather than an O-centered CBQ-O•.30 To the best of our knowledge, this is the first report that a C-centered ketoxy radical was characterized by the complementary application of ESR spin trapping, NMR and FTICR/MS methods.

4. The unequivocal identification of the radical form of the ketoxy radical adduct In the above study, a new ketoxy radical (•CBQ-OH, MW: 157) adduct with DMPO (DMPO-157) was observed for DCBQ/t-BuOOH system.27 However, although we can detect its nitroxide radical form DMPO-157 by ESR, we cannot



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directly observe the radical form by MS, only its corresponding nitrone form. Similarly, only the ESR-silent DMPO-157 nitrone adduct can be isolated by HPLC, but not its corresponding ESR-active nitroxide radical form.27 In summary, we were unable to get the clean DMPO-157 nitroxide radical adduct and thus observe its pure 6-line ESR spectra with no disturbance of other simultaneously-produced radicals. It might be due to the following reasons: either the DMPO-157 nitroxide radical adduct was too reactive to further oxidize to its ESR-silent nitrone form during HPLC analysis; or the radical adduct concentration was too low to be measured by ESR. Therefore, in a recent study,31 we tried to address these issues: Can we find a spin-trap which can produce a stable enough nitroxide radical adduct with the ketoxy radical; if so, can we characterize the radical form by MS directly; and can it be isolated and purified by semi-preparative HPLC, then detect its pure ESR signal? It has been found that certain radical adducts with 5-t-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO), a spin-trap possessing analogous DMPO structure, were much stable than their corresponding DMPO adducts.32 Thus, BMPO was selected for our study. To our surprise, the half-life for BMPO/•CBQ-OH radical adduct was found to be around 5 hours (only 15 min for DMPO/•CBQ-OH). The generation of BMPO/ketoxy radical derived nitroxide and nitrone adducts (here generally referred as BMPO-157) by DCBQ/t-BuOOH with BMPO were investigated preliminarily by ESI-Q-TOF-MS. As expected, 1-Cl isotope clusters at m/z 354, the nitrone form of BMPO-157, were detected by MS. By serendipity, a new peak with 1-Cl isotope clusters at m/z 355 was also detected concurrently, which might be the nitroxide radical form of BMPO-157 on the basis of theoretical calculations. For further confirmation, we tried to isolate the two distinct forms of BMPO-157 by HPLC. Because the nitroxide radical form of BMPO-157 is sensitive to acidic medium, we used neutral mobile phase instead. Two new peaks with the different retention times were detected. One of them was found to be the nitrone form of BMPO-157 (m/z 354), while the other its corresponding nitroxide radical form (m/z 355) (Fig. 6), which were further confirmed by MS/MS. These findings clearly


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indicated that the two forms of BMPO-157 could be characterized by HPLC-MS, and the peak with m/z at 355 should be the nitroxide radical form of BMPO-157, which was further confirmed to be the case by FTICR/MS and theoretical calculations. To further substantiate whether the peak at m/z 355 is the radical adduct BMPO/•CBQ-OH, it is imperative to get the pure fraction with the peak at m/z 355 with ESR examination. As expected, a pure 6-line ESR spectra could be detected with the isolated fraction by semi-preparative HPLC, corresponding to the six equal ESR peaks from DCBQ/t-BuOOH with BMPO (aH = 25.9; aN = 16.04; and aN/aH = 0.62) (Fig. 6). The above findings may also have important biological relevance. Our results indicate that these XQ may interact with hydroperoxides and exert toxic effects not only via facilitated generation of alkoxyl/hydroxyl radicals, but also via the production of ketoxy radicals which may have direct interaction with DNA, protein and lipids. Recently, H2O2 has been often used as an oxidizing agent for destruction of chlorinated

phenols

and

other

persistent

substances.4-6

organic

In

these

“environmentally-friendly” systems, mM levels of H2O2 have been often employed. One recent research demonstrated that 2,6-DCBQ could be observed quickly and then further

transformed

to

other

products

during

advanced

oxidation

of

2,4,6-trichlorophenol (2,4,6-TCP) by H2O2 and iron complexes.6 Another research found that H2O2 could enhance TCBQ hydrolysis rate hundreds of times.26 It was point out that H2O2-induced XQ decomposition mechanism may be critical in systems where H2O2 is either employed or generated. However, the exact molecular mechanisms underlying such further changes remain unclear yet. Our new discoveries may provide a new perspective to better elucidate such decomposition pathways during advanced oxidation treatment for waste water or remediation process in which XQ are produced.

5. Metal-independent decomposition of endogenous ROOH by XQ



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Lipid peroxidation (LPO) has been extensively investigated for many years.33-36 13-Hydroperoxy-9,11-octadecadienoic acid (13-HPODE) is one of the most well-known endogenous ROOH.33-36. In the presence of Fe(II), 13-HPODE could first produce lipid alkoxyl radical, which will further generate reactive lipid alkyl radicals and genotoxic aldehydes like 4-hydroxy-2-nonenal (HNE).37-40 We have shown above that alkoxyl/ketoxy radicals can be generated during the t-BuOOH decomposition by XQ metal-independently.22,27,31 Since 13-HPODE is a secondary ROOH possessing a long chain, while t-BuOOH is a tertiary ROOH with only a short t-butyl group, we presumed that the interactions between DCBQ and the two ROOH should not only have similarities, but also differences. Therefore, in a recent research,41 we tried to address these issues: 1) Could DCBQ and other XQ facilitate 13-HPODE breakdown and production of the reactive radical intermediates and the genotoxic aldehydes metal-independently; 2) If so, what are the differences: i) between the breakdown of 13-HPODE and t-BuOOH by DCBQ, and ii) between DCBQ- and Fe(II)-induced 13-HPODE decomposition; and 3) What is the underlying molecular mechanism? With POBN as spin-trap, the characteristic POBN adducts could be generated only when both DCBQ and 13-HPODE were present simultaneously. To differentiate different POBN adducts formed, HPLC/MS was employed to separate and identify them. Selective reaction monitoring (SRM) mode was used for analysis due to the low levels of spin-trapped radical adducts formed. The following three POBN/radical (pentyl,

7-carboxyheptyl,

1-(7-carboxyheptyl)-4,5-epoxy-2-decenyl,

1-(1,2-epoxyheptyl)-10-carboxy-2-decenyl

radical) adducts were

observed

or in

DCBQ/13-HPODE with POBN. As expected, CBQ-OH and a quinone-lipid alkoxyl conjugate CBQ(OH)-13-O-L were found as two major reaction products for DCBQ/13-HPODE. Serendipitously, a new product CBQ(OH)-9-O-LO was also detected. HNE was analyzed by GC/MS after derivatizations. We found that DCBQ could indeed enhance HNE formation. Analogous results were obtained when DCBQ was replaced by other XQ.


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Put the above findings together, the following molecular mechanism for DCBQ-induced 13-HPODE decomposition was suggested (Fig. 7): 13-HPODE (13-LOOH) may first attack DCBQ via nucleophilic substitution, generating a chloroquinone/peroxide intermediate CBQ-13-OOL, which may form 13-LO• and CBQ-O• via homolytical decomposition. CBQ-O• then either dismutates to produce CBQ-OH, or isomerizes to produce •CBQ=O, which then coupled with 13-LO• to form CBQ(OH)-13-O-L. The 13-LO• could either break down to pentyl radical via β-scission, or isomerize to produce the C-centered OL•, which then via oxygen addition to generate a new ROOH (9-OLOOH). 9-OLOOH then may interact with DCBQ to produce 9-OLO•, which either coupled with •CBQ=O to form CBQ(OH)-9-O-LO, or decompose to 7-carboxyheptyl radical via β-scission.

6. Conclusions and future research The above finding represent an unusual pathway for alkoxyl/ketoxy radical and/or HNE production not involving redox-active metals, and may explain, at least in part, the potential carcinogenicity of not only PCP, but also other widely used halogenated aromatic carcinogens (hexachlorobenzene, Agent Orange and the brominated flame-retardants), since these chemicals can be metabolized in vivo,3-11,42-45 or oxidized to tetra-, di- or mono-XQ. Our findings showed that XQ may interact with ROOH and cause deleterious effects via facilitated generation of alkoxyl/ketoxy radicals and/or HNE, and hence elevated damages to macromolecules. It should be emphasized that several issues need to be further investigated, particularly with regard to their biological relevance. For instance: How does the reaction of XQ/ROOH compare kinetically with reactions with other good nucleophiles like glutathione present at high concentrations in vivo? Could ketoxy radical be observed in cells or in an animal model? Could the suggested enoxy radical and quinone-peroxide intermediates be observed by a freeze-quenching technique? Could the new ketoxy radical react with macromolecules such as DNA, protein and lipids?



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Funding Sources The study was supported by the SPRP CAS (XDB01020300); NSFC (21237005, 21321004, 21477139, 21477139, 21407163); PFSC (2014M561078).

REFERENCES [1] Bolton, J. L., Trush, M. A., Penning, T. M., Dryhurst, G., and Monks, T. J. (2000) Role of quinones in toxicology. Chem. Res. Toxicol. 13, 135-160. [2] Song, Y., Wagner, B. A., Witmer, J. R., Lehmler, H. J., and Buettner, G. R. (2009) Nonenzymatic displacement of chlorine and formation of free radicals upon the reaction of glutathione with PCB quinones. Proc. Natl. Acad. Sci. USA 106, 9725-9730. [3] Zhu, B. Z., and Shan, G. Q. (2009) Potential mechanism for pentachlorophenol-induced carcinogenicity: a novel mechanism for metal-independent production of hydroxyl radicals. Chem. Res. Toxicol. 22, 969-977. [4] Meunier, B. (2002) Catalytic degradation of chlorinated phenols. Science 296, 270-271. [5] Gupta, S. S., Stadler, M., Noser, C. A., Ghosh, A., Steinhoff, B., Lenoir, D., Horwitz, C. P., Schramm, K. W., and Collins, T. J. (2002) Rapid total destruction of chlorophenols by activated hydrogen peroxide. Science 296, 326-328. [6] Sorokin, A., Meunier, B., and Seris, J. L. (1995) Efficient oxidative dechlorination and aromatic ring cleavage of chlorinated phenols catalyzed by iron sulfophthalocyanine. Science 268, 1163-1166. [7] Zhao, Y. L., Qin, F., Boyd, J. M., Anichina, J., and Li, X. F. (2010) Characterization and determination of chloro- and bromo-benzoquinones as new chlorination disinfection byproducts in drinking water. Anal. Chem. 82, 4599-4605. [8] Qin, F., Zhao, Y. Y., Zhao, Y. L., Boyd, J. M., Zhou, W. J., and Li, X. F. (2010) A toxic disinfection by-product, 2,6-dichloro-1,4-benzoquinone, identified in drinking water. Angew. Chem., Int. Ed. 49, 790-792. [9] Chignell, C. F., Han, S. K., Moulthys-Mickalad, A., Sik, R. H., Stadler, K., and Kadiiska, M. B. (2008) EPR studies of in vivo radical production by 3,3 ',5,5 '-tetrabromobisphenol A (TBBPA) in the Sprague-Dawley rat. Toxicol. Appl. Pharmacol. 230, 17-22. [10] Teuten, E. L., Xu, L., and Reddy, C. M. (2005) Two abundant bioaccumulated halogenated compounds are natural products. Science 307, 917-920. [11] Kelly, B. C., Ikonomou, M. G., Blair, J. D., Morin, A. E., and Gobas, F. A. P. C. (2007) Food web-specific biomagnification of persistent organic pollutants. Science 317, 236-239. [12] Halliwell, B., and Gutteridge, J. (2007) Free Radicals in Biology and Medicine, Oxford University Press, Oxford, U.K. . [13] Marnett, L. J. (2000) Oxyradicals and DNA damage. Carcinogenesis 21, 361-370. [14] Zhu, B. Z., Kitrossky, N., and Chevion, M. (2000) Evidence for production of hydroxyl radicals by pentachlorophenol metabolites and hydrogen peroxide: A metal-independent organic Fenton reaction. Biochem. Biophys. Res. Commun. 270, 942-946.


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[30] Li, A. S. W., and Chignell, C. F. (1991) The NoH value in EPR spin trapping: a new parameter for the identification of 5,5-dimethyl-1-pyrroline-N-oxide spin adducts. J. Biochem. Biophys. Methods 22, 83-87. [31] Huang, C. H., Shan, G. Q., Mao, L., Kalyanaraman, B., Qin, H., Ren, F. R., and Zhu, B. Z. (2013) The first purification and unequivocal characterization of the radical form of the carbon-centered quinone ketoxy radical adduct. Chem. Commun. 49, 6436-6438. [32] Zhao, H. T., Joseph, J., Zhang, H., Karoui, H., and Kalyanaraman, B. (2001) Synthesis and biochemical applications of a solid cyclic nitrone spin trap: A relatively superior trap for detecting superoxide anions and glutathiyl radicals. Free Radic. Biol. Med. 31, 599-606. [33] Yin, H. Y., Xu, L. B., and Porter, N. A. (2011) Free radical lipid peroxidation: Mechanisms and analysis. Chem. Rev. 111, 5944-5972. [34] Niki, E. (2009) Lipid peroxidation: Physiological levels and dual biological effects. Free Radic. Biol. Med. 47, 469-484. [35] Blair, I. A. (2008) DNA adducts with lipid peroxidation products. J. Biol. Chem. 283, 15545-15549. [36] Lee, S. H., Oe, T., and Blair, I. A. (2001) Vitamin C-induced decomposition of lipid hydroperoxides to endogenous genotoxins. Science 292, 2083-2086. [37] Qian, S. Y., Yue, G. H., Tomer, K. B., and Mason, R. P. (2003) Identification of all classes of spin-trapped carbon-centered radicals in soybean lipoxygenase-dependent lipid peroxidations of omega-6 polyunsaturated fatty acids via LC/ESR, LC/MS, and tandem MS. Free Radic. Biol. Med. 34, 1017-1028. [38] Iwahashi, H., Hirai, T., and Kumamoto, K. (2006) High performance liquid chromatography/electron spin resonance/mass spectrometry analyses of radicals formed in an anaerobic reaction of 9- (or 13-) hydroperoxide octadecadienoic acids with ferrous ions. J. Chromatogr. A 1132, 67-75. [39] Spiteller, P., Kern, W., Reiner, J., and Spiteller, G. (2001) Aldehydic lipid peroxidation products derived from linoleic acid. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 1531, 188-208. [40] Schneider, C., Porter, N. A., and Brash, A. R. (2008) Routes to 4-hydroxynonenal: Fundamental issues in the mechanisms of lipid peroxidation. J. Biol. Chem. 283, 15539-15543. [41] Qin, H., Huang, C. H., Mao, L., Xia, H. Y., Kalyanaraman, B., Shao, J., Shan, G. Q., and Zhu, B. Z. (2013) Molecular mechanism of metal-independent decomposition of lipid hydroperoxide 13-HPODE by halogenated quinoid carcinogens. Free Radic. Biol. Med. 63, 459-466. [42] Haugland, R. A., Schlemm, D. J., Lyons, R. P., Sferra, P. R., and Chakrabarty, A. M. (1990) Degradation of the chlorinated phenoxyacetate herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by pure and mixed bacterial cultures. Appl. Environ. Microbiol. 56, 1357-1362. [43] Koss, G., Losekam, M., Seidel, J., Steinbach, K., and Koransky, W. (1987) Inhibitory effect of tetrachloro-p-hydroquinone and other metabolites of hexachlorobenzene on hepatic uroporphyrinogen decarboxylase activity with reference to the role of glutathione. Ann. N. Y. Acad. Sci. 514, 148-159. [44] Vanommen, B., Adang, A. E. P., Brader, L., Posthumus, M. A., Muller, F., and Vanbladeren, P. J. (1986) The microsomal metabolism of hexachlorobenzene. Origin of the covalent binding to protein. Biochem. Pharmacol. 35, 3233-3238.


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[45] Czaplicka, M. (2004) Sources and transformations of chlorophenols in the natural environment. Sci. Total Environ. 322, 21-39.

Cl Cl

O

O

Cl

O

Cl

O

O

Cl Cl

O

TrCBQ

TCBQ

Cl

O

Cl

Cl

O

O

O

2,6-DCBQ

2-CBQ

Cl

Cl

2,3-DCBQ

Br Br

O

O

O

Cl

Cl

F

Br

F

Fig. 1: Chemical structures of the halogenated quinones


O

2,5-DCBQ

Br

TBBQ

Cl

O

O

F F

TFBQ


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

X X

O

HCl

X

+ O

X

Page 18 of 21

O

OOH

H2O2 Nucleophilic Substitution

X

TXBQ

X

O

X

TrXBQ-OOH

X = F, Cl, Br

Homolytic Decomposition

H2 O2

X

O

O

X

O

O

OH X

O

X

-

TrXBQ-O

X

O

X

+

OH

.

TrXBQ-O

Fig. 2: Proposed mechanism for •OH production by tetrahalo-1,4-benzoquinone (TXBQ, X = F, Cl, Br) and H2O2: a nucleophilic reaction may take place between TXBQ and H2O2, forming a trihalo-hydroperoxyl-1,4-benzoquinone (TrXBQ-OOH) intermediate, which can decompose homolytically to produce •OH and trihalo-hydroxy-1,4-benzoquinone radical (TrXBQ-O•). Reproduced with permission from Zhu, B. Z., Kalyanaraman, B., and Jiang, G. B. (2007) Molecular mechanism for metal-independent production of hydroxyl radicals by hydrogen peroxide and halogenated quinones. Proc. Natl. Acad. Sci. USA 104, 17575-17578.16 Copyright (2007) National Academy of Sciences, U.S.A.


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O Cl

DCBQ

O

Cl Cl

O

Cl

O

DCSQ•-

CH3

t-BuOOH

10 G

H3C C OOH CH3

DCBQ/t-BuOOH • * ? ¶

•* •

•* •

?

* ? ¶

• ?

DCHQ, 0.1 mM

DCHQ/MPO

* DMPO/t-BuO• • DMPO/•CH3 ¶ DMPO/•OCH3 ? Unidentified

Fig. 3: Metal-independent decomposition of t-BuOOH and formation of alkoxyl radicals by 2,5-dichloro-1,4-benzoquinone (DCBQ) or its reduced form 2,5-dichlorohydroquinone (DCHQ) in the absence or presence of myeloperoxidase. Reproduced with permission from Zhu, B. Z., Zhao, H. T., Kalyanaraman, B., Liu, J., Shan, G. Q., Du, Y. G., and Frei, B. (2007) Mechanism of metal-independent decomposition of organic hydroperoxides and formation of alkoxyl radicals by halogenated quinones. Proc. Natl. Acad. Sci. USA 104, 3698-3702.22 Copyright (2007) National Academy of Sciences, U.S.A.

O Cl

HCl Cl

+

O

Nucleophilic Substitution

O

Cl

DCBQ

O Cl

O

O

O

Radical Coupling

O-t-Bu

Cl

O

Spin Isomerization

O

Keto-Enol Tautomerization

DMPO

Cl

O

CBQ-OO-t-Bu

O

Homolytic Decomposition

•CBQ=O

O

OO-t-Bu

t-BuOOH

OH O-t-Bu

CBQ(OH)-O-t-Bu

Cl

O

O

O Dismutation Cl

O

O

O CBQ-O-

CBQ-O•

+

CH3 β -Scission

DMPO/CBQ=O

t-BuO DMPO

DMPO/t-BuO


+ CH3COCH3


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Fig. 4: Proposed mechanism for DCBQ-mediated t-BuOOH decomposition and formation of the novel carbon-centered quinone ketoxy radical and the final reaction product CBQ(OH)-O-t-Bu. Reproduced with permission from Zhu, B. Z., Shan, G. Q., Huang, C. H., Kalyanaraman, B., Mao, L., and Du, Y. G. (2009) Metal-independent decomposition of hydroperoxides by halogenated quinones: Detection and identification of a quinone ketoxy radical. Proc. Natl. Acad. Sci. USA 106, 11466-11471.27 Copyright (2009) National Academy of Sciences, U.S.A.

O Cl

O

Detected First Time

O

aH

= 28.8 G aN = 17.0 G aH/aN = 0.59

O

Spin Isomerization

O Cl

DCBQ/H2O2

*

*

?

?

O • CBQ-OH

*

?

? ?

? DMPO/• CBQ-OH

?

*

* DMPO/ • OH

Fig. 5: DMPO nitroxide adduct with the carbon-centered quinone ketoxy radical. Reproduced with permission from Zhu, B. Z., Shan, G. Q., Huang, C. H., Kalyanaraman, B., Mao, L., and Du, Y. G. (2009) Metal-independent decomposition of hydroperoxides by halogenated quinones: Detection and identification of a quinone ketoxy radical. Proc. Natl. Acad. Sci. USA 106, 11466-11471.27 Copyright (2009) National Academy of Sciences, U.S.A.

100 O O + Cl

O Cl

O

O O H N

Spin-Trapping

C O tBu O

O • CBQ-OH

O

O N

Cl

OH

BMPO

C O tBu O

BMPO/CBQ-OH Nitrone m/z 354

C O tBu O

Keto-Enol Tautomerization One-Electron Oxidation

OHO N

%

Cl

O

OH O N O H

Half Time 5 hr

0

355

MS 354 354

ESR

356 356

357 358 358

360

m/z

C O tBu O

BMPO/CBQ-OH Nitroxide m/z 355

BMPO/•CBQ-OH: aH = 26.5 G, aN = 16.28 G


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Fig. 6: The purification and unequivocal characterization of the radical form of the BMPO/ ketoxy radical adduct. Huang, C. H., Shan, G. Q., Mao, L., Kalyanaraman, B., Qin, H., Ren, F. R., and Zhu, B. Z. (2013) The first purification and unequivocal characterization of the radical form of the carbon-centered quinone ketoxy radical adduct. Chem. Commun. 49, 6436-6438.31 Reproduced by permission of The Royal Society of Chemistry. http://pubs.rsc.org/en/content/articlelanding/2013/cc/c3cc42732c

Reactive C-Radicals

Endogeneous Lipid Hydroperoxide

Carcinogenic Aldehyde

Fig. 7: Metal-independent decomposition of endogenous lipid hydroperoxide 13-HPODE by halogenated quinoid carcinogens. Reprinted from Free Radic. Biol. Med.; Volume 63; Qin, H., Huang, C. H., Mao, L., Xia, H. Y., Kalyanaraman, B., Shao, J., Shan, G. Q., and Zhu, B. Z.; Molecular mechanism of metal-independent decomposition of lipid hydroperoxide 13-HPODE by halogenated quinoid carcinogens; 459-466; Copyright 2013, with permission from Elsevier.41


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